PROJECT SUMMARY Antibiotics, in particular the cell wall-acting ?-lactams, were one of the greatest medical advances of the 20th century; however, they are quickly becoming obsolete due to antibiotic resistance that has been augmented by poor antibiotic stewardship and a void in novel antibiotic discovery. Despite recent renewed attention from the pharmaceutical industry, there continues to be a need for novel targets, compounds and approaches to treatment of bacterial infections, including the repurposing of existing drugs or pre-clinical compounds and the broad implementation of combination therapy. We and others have recently identified a family of highly conserved proteins collectively known as the PASTA (penicillin-binding and serine/threonine kinase associated) kinases as an exciting new target for the development of novel antimicrobials due to their central role in virulence, biofilm formation and ultimately ?-lactam resistance in a wide variety of important Gram positive pathogens including Staphylococcus aureus, Listeria monocytogenes, and Mycobacterium tuberculosis. S. aureus is a major cause of morbidity and mortality due to the variety of infections that it can cause, ranging from innocuous skin infections and boils to more severe chronic endocarditis, pneumonia, sepsis and ultimately death. S. aureus has a wide range of factors that allow it to colonize as a commensal organism, a major risk factor for development of subsequent infection in ~30% of the population. The lack of a vaccine, coupled with the recent expansion of antibiotic resistance, particularly methicillin resistance (Methicillin Resistant Staphylococcus aureus, MRSA) and more recently vancomycin resistance, among both community and hospital acquired strains has led to the understanding that we are in desperate need of new therapeutic options. In S. aureus the PASTA kinase, Stk1, is required for ?-lactam resistance. We have performed a screen and identified specific inhibitors of Stk1 that resensitize MRSA to ?-lactam antibiotics. Using orthogonal approaches, this proposal will identify the signaling cascades and Stk1 substrates that regulate ?-lactam resistance. We will utilize forward genetics, our novel kinase inhibitors and new genetic tools, in combination with classic genetic and biochemical approaches to understand the fundamental signaling cascades downstream of the PASTA kinase Stk1. Given the high level of conservation of the PASTA kinases and their signaling axes, the results from this work are likely to be broadly applicable to a wide variety of important Gram positive pathogens. Furthermore, these studies will characterize a mechanism of action for a series of kinase inhibitors currently in development as part of a novel combination therapy approach to MRSA infections.